As described above, our initial attempt to develop immunotherapy for pediatric sarcomas targeted the tumor specific fusion proteins generated by the t(11,22) and t(2,13) in Ewing's sarcoma and alveolar rhabdomyosarcoma respectively. While these targets are attractive for their specificity, they may not prove efficacious as a tumor antigen due to the limited number of epitopes available from the breakpoint region. Therefore, we are very interested in identifying new antigens and new approaches to immunize patients toward pediatric sarcomas. To this end, we have focused upon the human system, since murine peptide presentation may differ substantially from that observed with similar epitopes in humans. Furthermore, we now have available several sets of autologous T cells, autologous dendritic cells and autologous tumor derived from patients treated on our protocols. Therefore, the primary focus of this project was to generate primary responses to autologous tumor ex vivo using T cells harvested from patients with pediatric sarcomas. The experimental set up involves the utilization of a variety of approaches to immunize with whole tumor: apoptotic body ingestion, RNA transfection, tumor lysates, etc. Autologous T cells were collected by apheresis from patients with Ewing's sarcoma at the time of initial presentation or following disease recurrence. Using autologous CD40 ligand-matured DCs that were manipulated to ingest autologous apoptotic Ewing's sarcoma cells, followed by re-stimulation of autologous T cells with tumor cells alone, we generated MHC Class I-restricted, tumor-specific CTLs from 4/4 of patients tested. Surprisingly, stimulation with control autologous CD40 ligand-matured dendritic cells (which had not ingested apoptotic Ewing's sarcoma), followed by autologous tumor also induced tumor-specific, MHC Class I-restricted CTL responses in 4/4 patients. These results suggested that DC were capable of non-specifically expanding already primed Ewing's reactive T cells that were apparently circulating in patients with Ewing's sarcoma. Polyclonal stimulation using anti-CD3/anti-CD28 beads, followed by tumor cell stimulation did not induce tumor-specific responses. Phenotypic analysis showed that responding cells in these cultures were CD8+/CD28-/CD137+ (4-1BB+). These results suggest that patients with Ewing's sarcoma prime circulating T cells in vivo during the course of their disease and show that stimulation with CD40 ligand-matured dendritic cells followed by autologous tumor is capable of amplifying such memory CTL responses. Furthermore, the expansion of such tumor reactive CTL appears to require co-stimulation via alternative, non-CD28 pathways, potentially implicating CD137 (4-1BB) and its ligand. Further experiments are underway to definitely identify the critical factors required to promote the expansion of primed tumor-specific cytolytic T cells from patients with Ewing's sarcoma, and to identify the targets of these endogenous anti-tumor responses. These results have been submitted for presentation at the Cancer Vaccine Week in New York City, October 2000.